PROJECT SUMMARY The Hedgehog (Hh) pathway is the major target to treat medulloblastoma (MB), the most common pediatric brain tumor. Current treatment regimens bring life-long devastating side effects to survivors, generating a huge burden to the patients? family. The only FDA-approved Hh pathway inhibitor, vismodegib, transiently inhibits tumor growth, and drug resistance and tumor relapse quickly ensue. Hence, a therapeutic target that specifically inhibits Hh pathways with fewer side effects are critically needed. Our previous research revealed that PDE4D is a novel positive regulator of the Hh pathway. Current PDE4D small molecule inhibitors blocked the growth of MB in mouse model. PDE4D regulates the Hh pathway downstream of the vismodegib target. Therefore, inhibiting PDE4D is effective to block the growth of vismodegib-resistant tumors. However, all current PDE4D inhibitors block the catalytic activity of PDE4D in the entire cell, rendering toxicity that limits their clinical applications. This proposal aims to learn from numerous previous failures in the development of PDE4D inhibitors in pharmaceutical companies, and to develop new avenue of PDE4D inhibition by disrupting its localization to particular subcellular sites. The long-term goal of this application is to develop inhibitors that dislocate PDE4D3 from the centrosome. Such inhibitors will efficiently suppress Hh pathway with minimal interference with other PDE4D-related biological processes, thereby reducing the common side effects associated with current PDE4D small molecule inhibitors. The overall objective in this proposal is to identify the molecular basis underlying the specific regulation of the Hh pathway by PDE4D activity at the centrosome, and to provide proof-of-principle evidences for the inhibition of MB growth after dislodging PDE4D from the centrosome. The central hypothesis is that PDE4D3 is anchored to the centrosome to locally inhibit PKA activity, thereby specifically promoting the Hh pathway, and that removing PDE4D3 from the centrosome may specifically inhibit the Hh pathway while minimally interfering other pathways. This hypothesis is based on the previous understanding of PDE4D biology and the preliminary data obtained in the applicant?s laboratory. Guided by the strong preliminary data, this hypothesis will be tested by two aims: 1) Determine roles of centrosome-anchored PDE4D3 in the Hedgehog pathway and Hh-signaling dependent cell proliferation; and 2) Dislocate PDE4D3 from the centrosome and evaluate its impact on the Hh pathway and the growth of vismodegib-resistant tumors in mouse model. This approach is innovative because it highlights a new paradigm of Hh pathway regulation by a compartmentalized PDE4D activity at the centrosome, and points to a new therapeutic regimen based on protein-protein interaction. Its significance is underlined by the expectation that it will lead to new a therapeutic method with fewer side effects for pediatric patients with Hh-related brain tumor.